In the cytoplasm of eukaryotic cells, the presence of an insult, whether chemical, thermal, or in the form of misfolded or aggregated protein, triggers a complex biological response referred to as the heat shock response.1–3
This phenomenon is associated with expression of heat shock proteins (HSPs), which also function as molecular chaperones, and of proteins involved in the ubiquitin-proteasome pathway.
Impaired induction of the heat shock response may lead to a defective stress-induced synthesis of HSPs and potentially the accumulation of aggregated proteins.1–3
As a result, protein folding-related diseases may occur. Due to the very limited proliferation potential of neurons, the nervous system is most prone to such diseases, and the ultimate result is neurodegeneration. In neurons, toxicity caused by misfolded proteins may result from an imbalance between normal chaperone capacity and production of dangerous protein species.1–3
Therefore, increased chaperone expression can potentially suppress protein neurotoxicity, suggesting possible therapeutic strategies.4,5
Indeed, several studies have reported a reduction in cellular toxicity upon expression of Hsp70 and Hsp40 in neurodegenerative aggregation disease models of polyglutamine diseases, such as Huntington's disease, spinal and bulbar muscular atrophy, and several ataxias (SCA1–3).6–9
In various cellular models of Alzheimer's disease, increased levels of Hsp70 promoted tau solubility and tau binding to microtubules10
and inhibited the propensity of Aβ to aggregate.11
In Parkinson's disease models, directed expression of Hsp70 or pharmacologic HSP modulation prevented the neuronal loss caused by α-synuclein.12,13
An effect of Hsp70 in conferring protection to the presynaptic and postsynaptic termini in response to stress has also been reported.14
The neuroprotective effect of Hsp70 extends to astrocytes, where Hsp70 induction reduces apoptosis and necrosis by glucose and oxygen depravation.15,16
Altogether, in the diseased brain, Hsp70 induction may play a multi-faceted, protective role on the damaged neuronal protoplasm, on specialized synapses and on supporting astrocytes.
On the other hand, elevated Hsp70 expression, such as detected in cancer cells, facilitates the malignant phenotype.17–19
This effect derives from the ability of Hsp70 to inhibit key effectors of the apoptotic machinery, including the apoptosome, the caspase activation complex, and apoptosis-inducing factor. Hsp70 also plays a role in the proteasome-mediated degradation of apoptosis-regulating proteins.17–19
Elevated expression of Hsp70 appears to be high enough to control apoptosis, because downregulation of Hsp70 using antisense approaches increases the sensitivity of tumor cells to serum withdrawal and apoptosis inducing factor (AIF).20
Further, a reduction in endogenous Hsp70 levels in and of itself promotes in vitro
the apoptotic death of tumor cells derived from a wide variety of cancers, including breast, colon, prostate, hepatocellular carcinoma, and glioblastoma, while displaying no toxicity toward normal epithelia derived from breast or prostate, or toward fetal lung fibroblast.21–24
In cancer cells, Hsp70 also contributes to the Hsp90 chaperone machine, a protein complex with important roles in regulating the function of several onco-proteins.25,26
Overall, the Hsp70 protein is overexpressed in most cancer cells and is induced by other stresses, including anticancer drugs, yet these events occur as a result of a general stress response. In contrast, the protective functions of Hsp70 manifest in a manner that depends upon the specific wiring and function of apoptotic elements within a cell.27
The data presented above indicate that modulation of Hsp70 expression offers several therapeutic avenues to ameliorate a range of human diseases. In neurodegenerative diseases, in which Hsp70 induction may confer a protective advantage, induction of Hsp70 by pharmacological means is a potentially viable therapy. On the other hand, in cancer, downregulation of Hsp70 is a valuable strategy to induce apoptosis and a potential effective means to overcome tumor cell resistance.
To speed the rate at which modulators of Hsp70 expression can be evaluated and identified, a strategy that probes an increase or decrease in Hsp70 levels in neuronal or cancer models is needed. Here we describe a simple, cell-based assay to quantify Hsp70 protein levels in un-engineered cells. The method, which relies on whole-cell immunodetection of Hsp70 in human cells, utilizes a minimal number of cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. The assay has been developed for the microtiter plate format, which requires expenditure of minimal amounts of compound, thus making this an ideal platform for small molecule testing in low- to medium-throughput format. The assay also uses commercially available reagents and requires an ordinary plate reader with chemiluminescence capability; thus, our assay can be implemented in most research laboratories. This fast and reliable assay should greatly improve the speed with which novel Hsp70 protein expression inducers or inhibitors can be identified and evaluated.